16. THE FUNCTIONAL GENOME Flashcards
*What is functional genomics?
- Functional genomics is the study of how genes & the intergenic regions are responsible for processes
- Looks at gene functions & interactions
What does WGS & WEGS identify & why is it proof for pathogenesis?
- WGS & WEGS identifies candidate genes
- 15 -20,000 SNPs are reduced to several candidate genes
- The candidate genes are then checked for co-segregation amongst family members
- However, proof of pathogenesis is needed because the candidate genes aren’t causative. In vitro & in vivo methods can be used for proof of pathogenesis
Name 5 in vitro techniques for studying gene function
- In vitro techniques study microorganisms or biological samples outside their biological context such as a test tube or petri dish
1. Cell cultures
2. ShRNA
3. SiRNA
4. IPSCs
5. CRISPR (both in vivo & in vitro)
How can blood or tissue biopsies show whether a genetic variant affects a protein?
- Blood or tissue biopsies can be used to show whether a genetic variant affects a protein
- A Western blot analysis of samples can be used for comparison & can reveal any effects on protein
- However, biopsies are invasive & painful so cell cultures are preferred. Some genes of interest also don’t show up in blood
What is a cell culture?
- A cell culture involves removing cells from an organism or animal & subsequently growing them in manipulated conditions which are favourable
What are the advantages of cell cultures?
+ Cell cultures are a cheap, rapid & reproducible model for studying cells
+ It’s a good alternative to using animal models as fewer animals are used & therefore less ethical issues
+ There are many commercially available tissue specific cell lines, so these can be used in cell cultures
What are the disadvantages of cell cultures?
- Cells behave differently in a petri dish compared to inside the organism. It’s a 2D environment so some of the factors are missing
- Doesn’t stimulate the actual conditions inside an organism. E.g doesn’t consider signals from other tissues such as paracrine signals
- Cell cultures don’t provide any information about gene expression & function so we don’t know how the gene can affect other tissues
What is ShRNA?
- Short-hairpin RNA is an artificial molecule of RNA that can be used to silence gene expression
- It is vector based
How can ShRNA be used to knock out genes?
- ShRNA can be packaged into a plasmid & the expression can be controlled by RNA Polymerase II
- Once the RNA is transcribed, it can be transported out of the nucleus by the protein EXPORTIN 5
- It is then cleaved by a DICER
- The cleaved fragments then bind to RISC (RNA induced silencing complex)
- Leads to gene silencing
Give two ways in which the localisation of an encoded protein can be studied
- ANTIBODY STAINING
2. TRANSFECT CELLS WITH GFP TAGGED MUTATED GENE OR GENE OF INTEREST - for visualisation
What are IPSCs?
- Induced pluripotent stem cells are pluripotent stem cells that have been reprogrammed with TF from adult somatic cells
What are the steps for producing IPSCs?
- Take a skin biopsy
- Fibroblasts grown in culture
- Adult somatic cells can be reprogrammed with transcription factors
- Adult somatic cells can de-differentiate into IPSCs
- IPCs can be stimulated under conditions to differentiate into certain cells
What is SiRNA?
- Short-interfering RNA molecules have a similar method to ShRNA
- But SiRNAs are chemically synthesised & aren’t vector based so doesn’t use a plasmid
What is CRISPR & it’s steps?
- CRISPR can be carried out both in vivo & in vitro
- CRISPR = Clustered regularly interspaced short palindromic repeats
1. Guide RNA binds to Cas9 forming a complex
2. Cas9 then searches for a target sequence by binding to Protospacer adjacent motif (PAM - 2-3bp sequence downstream of guide RNA)
3. Cleavage of target sequence leads to knockout of the gene
4. Mutations can be introduced with Non-homologous end joining or Homology directed repair
Name 4 in vivo techniques for studying gene function
- Animal studies - mouse/zebrafish
- Morpholinos
- CRISPR
- ENU screens
Give 4 reasons why mice are used as the mammalian model for human genetics?
+ Mice have an accelerated lifespan where 30 years = 1 human year, so their whole lifespan can be studied in a few years
+ Mice are small & reproduce quickly so they are easy to study
+ Mice are genetically similar to humans so they are likely to be affected by similar diseases
+ Using mice is more ethical than using larger animals or non-human primates
How can mice be used to introduce mutations?
- A targetting vector can be constructed & inserted into the nucleus of embryonic pluripotent stem cells
- The targetting vector will contain a DNA cassette which is flanked upstream or downstream producing homology arms
- The casette replaces the knockout gene & the cells can be grown to blastocysts which can then be implanted into mice
*Give three reasons for why zebrafish are a good human model for human genetics?
- Zebrafish are cheap to use
- Zebrafish develop in 5 days
- Zebrafish produce many eggs which are optically transparent & they grow outside the mother’s body (ex vivo), making it easy to study them
How can morpholinos be used to knockout genes in zebra fish?
- Morpholinos (MO) are nucleotide analogs that recognise & bind to short sequences at the transcription or splicing site
What are two types of morpholinos?
- TRANSLATION BLOCKING MOs - target the transcription site
2. SPLICE INHIBITING MOs - target the splice site
